scholarly journals Cloning of the spoT Gene of “CandidatusPhlomobacter fragariae” and Development of a PCR-Restriction Fragment Length Polymorphism Assay for Detection of the Bacterium in Insects

2000 ◽  
Vol 66 (8) ◽  
pp. 3474-3480 ◽  
Author(s):  
Xavier Foissac ◽  
Jean-Luc Danet ◽  
Leyla Zreik ◽  
Jeanne Gandar ◽  
Jean-Georges Nourrisseau ◽  
...  
Keyword(s):  
Restriction Fragment ◽  
Fragment Length ◽  
Rrna Gene ◽  
Reading Frame ◽  
Length Polymorphisms ◽  
Spot Gene ◽  
The Many ◽  
The 16S Rrna Gene ◽  
Pcr Test ◽  
Restriction Fragment Length

ABSTRACT Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium “CandidatusPhlomobacter fragariae” is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between “P. fragariae” and other insect-associated proteobacteria, isolation of “P. fragariae” genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from “P. fragariae”-infected strawberry plants. It encodes part of a “P. fragariae” open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish “P. fragariae” from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry “P. fragariae.” Interestingly however, the “P. fragariae”spoT sequence could be easily detected in whiteflies proliferating on “P. fragariae”-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.

scholarly journals Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces

2003 ◽  
Vol 69 (2) ◽  
pp. 1251-1262 ◽  
Author(s):  
Koji Nagashima ◽  
Takayoshi Hisada ◽  
Maremi Sato ◽  
Jun Mochizuki
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rrna Gene ◽  
The 16S Rrna Gene ◽  
Restriction Fragment Length

ABSTRACT New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.

Polymorphisms in the nuclear DNA of Achlya species: some taxonomic implications

1989 ◽  
Vol 35 (12) ◽  
pp. 1146-1155 ◽  
Author(s):  
J. Stephen Horton ◽  
Paul A. Horgen
Keyword(s):  
Southern Blot ◽  
Restriction Fragment ◽  
Ribosomal Dna ◽  
Fragment Length ◽  
Restriction Fragment Length Polymorphisms ◽  
Dna Polymorphisms ◽  
Rrna Gene ◽  
Cdna Clones ◽  
Length Polymorphisms ◽  
Restriction Fragment Length

Within the genus Achyla, which belongs to the class of fungi known as the Oomycetes, taxonomic judgments have traditionally been made using a variety of sexual criteria. We have used restriction fragment length polymorphisms as a new taxonomic character to examine intra- and inter-specific variation within this genus. Using a cDNA clone coding for the Achlya 18S rRNA gene as a hybridization probe, a 10-kb fragment of ribosomal DNA (rDNA) from Achlya ambisexualis strain E87 was cloned and then mapped for selected restriction enzyme sites. In Southern blot hybridizations, both this rDNA fragment and cloned 18S cDNAs revealed differences in the rDNA organization of A. ambisexualis E87 (male) and a female isolate of A. ambisexualis strain 734. No differences in the rDNAs were detected between the two heterothallic isolates A. ambisexualis E87 and A. bisexualis 65-1. Southern blot hybridizations suggested that two different rDNA organizations may exist within the genome of the homothallic strain A. heterosexualis B14. cDNA clones coding for two different hormonally regulated genes revealed the same relationships between the four isolates studied as those determined with rDNA probes. Two homothallic Achlya strains recently isolated from nature were found to have additional DNA polymorphisms not detected in the laboratory strains. Phenetic analysis distinguished the same similarities that were evident upon inspection of the hybridization data. Taken together, these data suggest different relationships between the isolates examined than do the previous taxonomic criteria by which species have been delimited within this genus.Key words: Achlya; restriction fragment length polymorphisms; ribosomal DNA; taxonomy.

scholarly journals Rapid Identification of Campylobacter,Arcobacter, and Helicobacter Isolates by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene

1999 ◽  
Vol 37 (12) ◽  
pp. 4158-4160 ◽  
Author(s):  
Stephen M. Marshall ◽  
Pasquale L. Melito ◽  
David L. Woodward ◽  
Wendy M. Johnson ◽  
Frank G. Rodgers ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rrna Gene ◽  
The 16S Rrna Gene ◽  
Restriction Fragment Length

A rapid two-step identification scheme based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was developed in order to differentiate isolates belonging to theCampylobacter, Arcobacter, andHelicobacter genera. For 158 isolates (26 reference cultures and 132 clinical isolates), specific RFLP patterns were obtained and species were successfully identified by this assay.

scholarly journals The application of genotyping techniques to the epidemiological analysis ofCampylobacter jejuni

1996 ◽  
Vol 117 (2) ◽  
pp. 233-244 ◽  
Author(s):  
C. J. Jackson ◽  
A. J. Fox ◽  
D. R. A. Wareing ◽  
D. N. Hutchinson ◽  
D. M. Jones
Keyword(s):  
Restriction Fragment ◽  
Fragment Length ◽  
Restriction Fragment Length Polymorphisms ◽  
Rrna Gene ◽  
Epidemiological Typing ◽  
Length Polymorphisms ◽  
Genotypic Analysis ◽  
Ofcampylobacter Jejuni ◽  
Reference Strains ◽  
Restriction Fragment Length

SummaryCampylobacter jejuniserogroup reference strains and collections of sporadic and outbreak- associated isolates were examined for restriction fragment length polymorphisms (RFLPs), usingC. jejunirandom chromosomal and 16S rRNA gene probes. A collection of 48 Penner (HS) and 14 Lior (HL) serogroup reference strains, plus 10 clinical isolates, generated 35 RFLP and 26 ribotype patterns. In combination the two loci generated 48 distinct genotypes. Both probes were able to differentiate between certain random isolates of the same HS/HL serogroups but greater discrimination was obtained with RFLP than with ribotyping. Genotyping distinguished accurately between related and unrelated strains when applied to several outbreaks. Genotypic analysis ofC. jejuniby restriction fragment length polymorphisms is a valuable technique for epidemiological typing. Chromosomal variation detected by the two unlinked probe loci provides some information about the genetic relationship between isolates.

Sign in / Sign up

Export Citation Format

Share Document